Human cytosolic 3alpha-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3beta-hydroxysteroid dehydrogenase activity: implications for steroid hormone metabolism and action.

Stephan Steckelbroeck,Yi Jin,Sridhar Gopishetty,Trevor M Penning,Busola Oyesanmi

doi:10.1074/jbc.m313308200

Abstract

The source of NADPH-dependent cytosolic 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5alpha-dihydrotestosterone (DHT) into inactive 3beta-androstanediol (3beta-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3alpha-/17beta-/20alpha-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3alpha- and 3beta-Diol (established by (1)H NMR spectroscopy). The rates of 3alpha- versus 3beta-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3alpha[17beta]-hydroxysteroid oxidase activity with 3alpha-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3beta-HSD/Delta(5-4) ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3alpha- and 3beta-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3alpha/3beta-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3alpha/3beta-HSD activities are achieved. The observation that AKR1Cs are a source of 3beta-tetrahydrosteroids is of physiological significance because: (i) the formation of 3beta-Diol (in contrast to 3alpha-Diol) is virtually irreversible, (ii) 3beta-Diol is a pro-apoptotic ligand for estrogen receptor beta, and (iii) 3beta-tetrahydrosteroids act as gamma-aminobutyric acid type A receptor antagonists.

Highlights

The source of NADPH-dependent cytosolic 3␤-hydroxysteroid dehydrogenase (3␤-HSD) activity is unknown to date
⌬5-3␤-hydroxysteroid into ⌬4-3-ketosteroids is catalyzed by two members of the short chain dehydrogenase/reductase (SDR) family: the bifunctional 3␤-HSD/⌬5–4 ketosteroid isomerase (3␤-HSD/KSI) isoforms type 1 and type 2, which express 3␤HSD and isomerase activity in a single protein [1, 2, 23]
To further address the substrate specificities of these isoforms, we developed an improved TLC system, which permitted the separation of authentic DHT, androstanedione,androsterone, 3␣-Diol, and 3␤-Diol (Fig. 2C)

Summary

IMPLICATIONS FOR STEROID HORMONE METABOLISM AND ACTION*

Human hepatoma (HepG2) cells (which lack 3␤-HSD/⌬5–4 ketosteroid isomerase mRNA expression, but express AKR1C1–AKR1C3) were able to convert DHT into 3␣and 3␤-Diol This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3␣/3␤-HSD activities of the AKR1C enzymes. ⌬5-3␤-hydroxysteroid into ⌬4-3-ketosteroids is catalyzed by two members of the short chain dehydrogenase/reductase (SDR) family: the bifunctional 3␤-HSD/⌬5–4 ketosteroid isomerase (3␤-HSD/KSI) isoforms type 1 and type 2, which express 3␤HSD and isomerase activity in a single protein [1, 2, 23] Besides their essential role in the formation of active steroid hormones, the 3␤-HSD/KSI isoforms catalyze the NAD(H)-dependent in vitro interconversion of 3-keto- and 3␤-hydroxy-5␣-androstanes [24, 25]. The human AKR1C isozymes are both nonpositional and non-stereo-selective HSDs and represent a potential cytosolic source of 3␤-tetrahydrosteroids in target tissues such as prostate and brain

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION